Preparation of Selenocysteine-Containing Forms of Human SELENOK and SELENOS

Abstract

Selenoprotein K (SELENOK) and Selenoprotein S (SELENOS) are the members of the endoplasmicreticulum-associated degradation (ERAD) complex, which is responsible for translocating misfolded proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Besides its involvement in the ERAD, SELENOK was shown to bind and stabilize the palmitoyl transferase DHHC6, and thus contributes to palmitoylation. SELENOK and SELENOS reside in the ER membrane by the way of a single transmembrane helix. Both contain an intrinsically disordered region with a selenocysteine (Sec) located one or two residues away from the C-terminus. Here, we describe the preparation of the Seccontaining forms of SELENOS and SELENOK. SELENOK, which contains no native cysteines, was prepared in an E. coli cysteine auxotroph strain by exploiting the codon and the insertion machinery of Cys for the incorporation of Sec. In contrast, the preparation of SELENOS, which contains functionally important cysteine residues, relied on E. coli’s native Sec incorporation mechanism.

Selenoprotein K Increases Efficiency of DHHC6 Catalyzed Protein Palmitoylation by Stabilizing the Acyl-DHHC6 Intermediate

“…The possibility that SELENOK functions to stabilize the acyl-DHHC6 intermediate and prevent hydrolysis before the acyl group can be transferred to a target protein highlights the importance of this step in the palmitoylation reaction that physiologically occurs on the cytosolic side of the ER membrane. Synthesis of SELENOK requires sufficient dietary selenium and dedicated translational machinery to insert the 21st amino acid into position 92 [ 1 , 3 , 18 ]. Exactly how SELENOK uses its Sec residue to stabilize the acyl-DHHC6 intermediate was not determined in this study.…”

“…The gel was then stained using Coomassie Brilliant Blue (Thermo Fisher Scientific, Waltham, MA, USA) per manufacturer’s instructions to validate equivalent quantities of SELENOK as well as equivalent DHHC6 pull-down. Western blots were performed and analyzed using an Odyssey scanner (Li-Cor, Lincoln, NE, USA) as previously described [ 18 ].…”

Impact of Selenium on Biomarkers and Clinical Aspects Related to Ageing. A Review

“…Ageing is related to changes in the renewal of proteins and mitochondria, and the aggregation of misfolded proteins seems to be a central feature in ageing and in age-related diseases, such as Alzheimer's disease and type 2 diabetes [31,32]. Several selenoproteins, i.a., selenoproteins F, K, S and T, which reside in the endoplasmic reticulum (ER), appear to participate in the control and removal of misfolded proteins and in the protection against ER stress, also including control of calcium homeostasis [4,5,33]. Hence, adequate selenium status may play a role in healthy ageing by these mechanisms also.…”

“…The trace element selenium (Se), as a significant essential nutritional factor, may remodel biochemical and physiological changes accompanying ageing by improving immune functions, mediating metabolic homeostasis and antioxidant defense, and also in the removal of misfolded proteins [4][5][6]. Se deficiency in ageing populations seems to increase Biomolecules 2021, 11, 1478 2 of 14 the risk of developing age-related diseases [7,8].…”